CN1147323C - 壳聚糖/明胶网络支架材料 - Google Patents
壳聚糖/明胶网络支架材料Info
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- CN1147323C CN1147323C CNB001062875A CN00106287A CN1147323C CN 1147323 C CN1147323 C CN 1147323C CN B001062875 A CNB001062875 A CN B001062875A CN 00106287 A CN00106287 A CN 00106287A CN 1147323 C CN1147323 C CN 1147323C
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- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种用于生物医学的壳聚糖/明胶网络支架材料及其制备方法，采用相分离法制备的壳聚糖/明胶多孔支架材料，材料价格低廉，生物相容性好，亲/疏水平衡性可调，适用于软骨组织细胞附着生长和不同类型细胞的需要，可避免交联剂产生的细胞毒副作用。同时保证了多孔材料的力学强度和生物(细胞)亲和性。
Description
技术领域
本发明属于生物医学工程。
技术背景
组织工程学是运用工程科学与生命科学的基本原理和方法，研究与开发生物学替代物来恢复、维持和改进组织功能的一门新兴学科。其基本思路是：在体外分离、培养细胞，将一定量的细胞种植到具有一定形状的三维生物材料支架内，并加以持续培养，最终形成具有一定结构的组织和器官并回植体内达到修复或重建的目的。壳聚糖为一种天然聚阳离子多糖(pKa＝6.3)。它具有良好的生物相容性且可生物降解，在生物医学方面应用广泛，如用作透析膜、伤口愈合促进剂及药物释放载体等。医用壳聚糖可以有效减轻关节软骨的退变，降低关节手术后创伤性关节炎的发病率。明胶为胶原蛋白的变性衍生物，而胶原蛋白为细胞外基质的重要成分，缓冲挤压的关节软骨的基质主要由II型胶原组成。明胶的结构单元为甘氨酸、脯氨酸和羟脯氨酸与赖氨酸(Gly-Pro-Hyp-Hyl)，系柔性两性电解质，等电点pHiso＝4.7。以往的工作多以合成材料，尤其是聚酯类为基材作支架材料，如聚乳酸(PLA)、聚乙醇酸(PGA)等，它们缺乏细胞结合位点[Rouhi A M.Contemporary Biomaterials，Understanding Surfaces is Key to the Design of Clinically Useful Materials，Science/Technology，1998，18：51-59]，且其降解产物呈酸性，产物的pH脉冲作用在一定程度上造成无菌炎症[Athanasiou K A，Niederauer G G，Agrawal C M，Sterilization，Toxicity，Biocompatibility andClinical Applications of Polylactic Acid/Polyglycolic Acid Copolymers，Biomaterials，1996，17：93-102]。曾有报道采用壳聚糖或壳聚糖/胶原、壳聚糖/硫酸软骨素为支架材料。虽然壳聚糖本身即有对软骨等器官生长的促进作用[Lu J X，Prudhommenaux F，Meunier A，Sedel L，Guillemin G，Effects of Chitosan on Rat Knee Cartilages，Biomaterials，1999，20：1937-1944]，但壳聚糖本体结构单一，可调节性差，不能满足细胞特异性要求[Madihally S V，Matthew HW T，Porous Chitosan Scaffolds for Tissue Engineering，Biomaterials，1999，20：1133-1142]。而胶原不但较明胶昂贵许多，其抗原性问题一直是学者争议的焦点。文献也证实壳聚糖/硫酸软骨素复合体支架作为载体，其性能并不优于壳聚糖多孔材料[Denuziere A，Ferrier D，DamourO，Domard A，Chitosan-Chondroitin Sulfate and Chitcsan-Hyaluronate PolyelectrolyteComplexes：Biological Properties，Biomaterials，1998，19：1275-1285]。明胶从胶原水解而得，很大程度上保留了胶原的生物相容性等优点，无抗原性。明胶亦被用作组织工程用支架材料，但其强度太低，难于实际应用[Kang H W，Tabata Y，Ikada Y，Fabrication of Porous GelatinScaffolds for Tissue Engineering，Biomaterials，1999，20：1339-1344；Choi Y S，Hong S R，Lee YM，Song K W，Park M H，Nam Y S，Studies on Gelatin-Containing Artificial Skin：II.Preparation and Characterization of Cross-Linked Gelatin-Hyaluronate Sponge，J Biomed MaterRes(Appl Biomater)，1999，48：631-639]。
本发明的目的是采用细胞相容性良好的天然可降解生物材料制备组织工程用细胞培养支架，且亲/疏水平衡等性质可调，避免交联剂产生的细胞毒性作用，能够适应不同类型细胞的需要。
发明内容
本发明采用壳聚糖和明胶为基材制备组织工程用多孔支架材料。制备步骤如下：
配制0.5～10ml 0.5～10％wt(优选2～7％wt)的壳聚糖/明胶水溶液，将溶液转移至与溶液不反应且耐低温的平底容器中形成1～10mm高度的液面，置入-30℃～70℃的低温冰箱中预冻8～24小时；将冷冻干燥机干燥室温度降至-30℃～-40℃，将培养皿移入干燥室；温度平衡后密闭干燥室开启抽气泵，维持干燥室气压小于5000帕斯卡24～72小时，然后升至常压后加热至40℃热烘1～3小时，即得多孔材料粗产品。粗产品精制过程如下：以5～20％氢氧化钠溶液洗涤多孔基材；用去离子水清洗5～10次；采用5％戊二醛溶液在50℃下浸泡2～5小时进行交联；常温下用2％硼氢化纳溶液浸泡进行脱醛处理；用去离子水冲洗5～10次；降低冻干机温度至-40℃，将多孔基材连同培养皿一起移入冻干机，抽真空至干燥室气压小于5000帕斯卡并保存8～24小时；将多孔基材取出后采用γ-射线进行2～10小时，累计10～80万拉德剂量的照射以达到灭菌；用聚乙烯薄膜将多孔基材密封。
本发明的壳聚糖/明胶网络支架材料价格低廉，生物相容性好，亲/疏水平衡性可调，能够适应不同类型细胞的需要，同时保证了多孔材料的力学强度和生物(细胞)亲和性。该支架材料非常适于软骨细胞附着生长。
具体实施方式
取精制后的脱乙酰化度80％壳聚糖2g和明胶2g放入80ml浓度2％的醋酸水溶液中，40℃下静置4小时，达完全溶解将溶液转移至100ml容量瓶，添加醋酸溶液，得到100ml壳聚糖/明胶溶液(A)。取液(A)20ml转移至90mm聚苯乙烯培养皿，置入-70℃低温冰箱中预冻24小时；控制冷冻干燥机干燥室温度至-40℃，将含预冻体的培养皿移入干燥室；温度平衡后密闭干燥室并开启抽气泵，维持干燥室气压小于50μatm帕斯卡并保持48小时：减压取出后40℃下热烘2小时，初步获得多孔材料(B)。取基材(B)浸入10％的氢氧化钠溶液中和多孔基材中的醋酸；用去离子水反复清洗，除去残留醋酸根等离子；将基材浸入0.5％戊二醛溶液交联5分钟；用2％硼氢化纳溶液浸泡30分钟脱醛；用去离子水反复清洗；置入冻干机进行复冻，采用Co-60辐射灭菌后密封保存，即得到组织工程用壳聚糖/明胶网络支架材料。
Claims (1)
1.一种壳聚糖/明胶网络支架材料，其特征在于配制0.5～10％wt的壳聚糖/明胶水溶液，将溶液转移至与溶液不发生反应且耐低温的平底容器中形成1～10mm高度的液面，置入-30℃～-70℃的低温冰箱中预冻8～24小时，将培养皿移入温度为-30℃～-40℃的冷冻干燥机干燥室中，温度平衡后密闭干燥室开启抽气泵，维持干燥室气压小于5000帕斯卡24～72小时，然后升至常压后加热至40℃热烘1～3小时，即得多孔材料粗产品。以5～20％氢氧化钠溶液洗涤该多孔基材粗产品，用去离子水清洗5～10次，再用5％戊二醛溶液在50℃下浸泡2～5小时进行交联，常温下用2％硼氢化纳溶液浸泡进行脱醛处理，再用去离子水冲洗5～10次，降低冻干机温度至-40℃，将多孔基材连同培养皿一起移入冻干机中，抽真空至干燥室气压小于5000帕斯卡并保持8～24小时，多孔基材取出后采用γ-射线进行2～10小时，累计10～80万拉德剂量的照射以达到灭菌，然后用聚乙烯薄膜将多孔基材密封。
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| CNB001062875A CN1147323C (zh) | 2000-05-09 | 2000-05-09 | 壳聚糖/明胶网络支架材料 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393369C (zh) * | 2006-09-20 | 2008-06-11 | 天津大学 | 明胶、蒙脱土和壳聚糖多孔组织工程支架材料及制备方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1327911C (zh) * | 2003-10-22 | 2007-07-25 | 曹谊林 | 组织工程表皮替代物及其制备方法 |
| FR2917625B1 (fr) * | 2007-06-20 | 2009-09-18 | Perouse Soc Par Actions Simpli | Traitement de dispositifs medicaux implantables resistants a la calcification |
| CN101327336B (zh) * | 2008-07-22 | 2012-06-27 | 厦门大学 | 一种骨/软骨组织工程支架材料及其制备方法 |
| CN101721748B (zh) * | 2009-11-25 | 2013-04-10 | 南京大学 | 双基因活化的骨-软骨复合移植体及其制备方法和应用 |
| CN101773683B (zh) * | 2010-03-03 | 2012-10-31 | 天津大学 | 壳聚糖修饰的海藻酸盐水凝胶三维多孔支架及其制备方法 |
| CN102488928A (zh) * | 2011-12-26 | 2012-06-13 | 重庆科技学院 | 一种碳纤维增强生物复合支架材料的制备方法 |
| CN103751832A (zh) * | 2014-01-10 | 2014-04-30 | 青岛海蓝生物制品有限公司 | 一种壳聚糖弹性多孔材料的制备方法 |
| CN103961748A (zh) * | 2014-05-13 | 2014-08-06 | 大连理工大学 | 一种动态构建ADSCs-壳聚糖/明胶水凝胶工程化软骨的方法 |
| CN103977448B (zh) * | 2014-05-21 | 2016-02-24 | 福建师范大学 | 一种不对称壳聚糖纳米纤维多孔膜的制备方法 |
| CN104721881B (zh) * | 2015-02-12 | 2016-09-14 | 宁波市医疗中心李惠利医院 | 一种高强度可降解软骨组织工程支架及其制备方法 |
| CN104927071B (zh) * | 2015-05-19 | 2017-05-17 | 武汉纺织大学 | 一种高取向多糖纤维膜的制备方法 |
| CN106913903A (zh) * | 2017-04-20 | 2017-07-04 | 李丹荣 | 一种负载生物活性玻璃微粒皮肤再生材料的制备方法 |
| CN114028613A (zh) * | 2021-10-21 | 2022-02-11 | 潍坊医学院 | 一种功能性骨修复复合支架、制备方法及应用 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393369C (zh) * | 2006-09-20 | 2008-06-11 | 天津大学 | 明胶、蒙脱土和壳聚糖多孔组织工程支架材料及制备方法 |
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| CN1272384A (zh) | 2000-11-08 |
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